APOE (Apolipoprotein E) is part of APOE/APOC gene cluster on chromosome 19 and codes for 3 protein isoforms ? APOE2, APOE3 and APOE4. APOE transports cholesterol and phospholipids in the periphery and brain. APOE isoforms differ in their lipid and receptor binding capacity and their role is associated with clearance of LDL, VLDL and chylomicrons. The inheritance of APOE4 allele increases and APOE2 decreases the risk for late onset AD (LOAD), but the mechanism is poorly understood. While APOE is involved in critical cellular functions such as oxidative processes, inflammation, glial cell and neuronal homeostasis, none of those can be dissociated from isoform specific binding, transport and delivery of cholesterol and phospholipids to different cell types. Recent reports suggest there might be a differential APOE-isoform specific effect on microglia mediated phagocytosis and function of immune receptors expressed in brain. Our preliminary data demonstrate APOE isoforms influence expression of immune receptors Dectin-1/Clec7a, Siglec1, Siglech, Oscar - involved in inflammatory response and phagocytosis. A strong support to an interconnected role for APOE and immune receptor mediated phagocytosis are our results of Multi-Dimensional Mass Spectrometry Shotgun Lipidomics of brain samples from AD patients, where we find significant differences in phospholipid molecular speciation in major phospholipid classes. We hypothesize the APOE isoform-specific effects on phagocytosis are driven by the different phospholipid composition of APOE lipid particles and/or by the differential effect of APOE isoforms on microglial transcriptome. We are proposing 3 Specific Aims to test the hypothesis: Aim 1. Establish isoform- dependent effect of APOE?2 allele on brain parenchymal and mitochondrial lipidome and transcriptome in AD patients and non-demented controls. The goal is to integrate lipid and transcriptional profiles and to reveal APOE allele controlled phenotypes and lipid molecular species for additional functional assays in SA3. Aim 2. Determine isoform- and age-dependent effect of APOE genotype on brain lipidome and transcriptome in mice expressing human APOE2, APOE3 or APOE4 isoforms. We will determine phospholipid content of native APOE in Astrocyte Conditioned media, lipid particles in brain interstitial fluid (ISF) and differences in brain parenchyma and mitochondrial lipidomes between mice expressing APOE2, APOE3 or APOE4 isoforms at 2 different ages. Aim 3. To test the effect of APOE-containing nanoparticles on A? phagocytosis in vitro and in vivo. The goals are to apply the knowledge about differences in lipid compositions of AD and mouse brains and to test the effect of phospholipid molecular species on microglia mediated A? phagocytosis and clearance of A? oligomeric species in in vitro and in vivo experimental systems.